Quick Links

How would you like to share?

From a neuron’s perspective, Alzheimer disease is looking more and more like a nasty case of indigestion. So suggests a new study that strengthens the link between AD and autophagy, a key mechanism by which cells chew up damaged organelles and protein waste. Publishing in the May 22 online Journal of Clinical Investigation (JCI), researchers led by Tony Wyss-Coray at Stanford University, Palo Alto, California, report that beclin 1, a key regulator of the autophagic pathway, is reduced in affected brain areas of AD patients. In addition, the scientists showed that in mice beclin 1 regulates the buildup of amyloid-β (Aβ) and that boosting autophagy with a beclin 1-expressing lentivirus reduces Aβ pathology. These findings suggest that targeting beclin 1 activity could hold promise as a novel therapeutic approach for AD.

“This study is the first to show that altering a known constituent of the autophagy pathway accentuates Alzheimer pathology,” said Ralph Nixon, New York University School of Medicine. Nixon was not involved with the new work but in a recent review (Nixon et al., 2008) champions the idea that impaired intracellular trafficking underlies a host of neurodegenerative disorders, including AD. (See a cartoon showing how beclin 1 fits into the autophagic-lysosomal trafficking scene.)

For Wyss-Coray, the hunch that autophagy—and beclin 1 in particular—could play a role in AD came from a curious source: the Lurcher mouse. Lurcher heterozygotes develop muscle coordination problems due to neurodegeneration in the cerebellum that derives from a spontaneous, semi-dominant glutamate receptor mutation (Zuo et al., 1997). Six years ago, a Neuron paper (Yue et al., 2002) by Nathaniel Heintz and colleagues at Rockefeller University, New York, identified the autophagy regulator beclin 1 as one of two proteins mediating the nervous system defects in Lurcher mice. That paper led Wyss-Coray to literature on an emerging connection between autophagy and neurodegeneration. At the time, he said, this link had not been explored using genetic models.

Intrigued by beclin 1’s involvement in the Lurcher phenotype and wondering whether the autophagy protein might also contribute to AD, Wyss-Coray approached Beth Levine at the University of Texas Southwestern Medical Center, Dallas, about her beclin 1 knockout mice. Levine was not only interested in using her mice to pursue the beclin 1-AD connection but also brought in another collaborator—Scott Small at Columbia University in New York. In microarray analyses, Small and colleagues had found decreased levels of beclin 1 RNA in the brains of AD patients.

The gene array findings spurred Wyss-Coray, first author Fiona Pickford, and colleagues to look at beclin 1 protein levels in AD. Sure enough, beclin 1 was dramatically reduced in AD brain tissue relative to controls. And as luck would have it, Eliezer Masliah, University of California at San Diego, was seeing the same thing in his studies of autophagy in Parkinson disease, for which he was using AD brains as controls. With Masliah joining Levine and Small as collaborators on the JCI work, the stage was set to unravel what now appears to be a much more complicated story. “This paper went through many incarnations,” Wyss-Coray told this reporter.

The JCI work adds a twist to earlier studies by Nixon and colleagues (Yu et al., 2005; Nixon et al., 2005) that were among the first to bring autophagy to the attention of the AD field. Using immunolabeling and electron microscopy to examine brain tissue from AD patients and AD transgenic mice (PS1/APP), the 2005 papers showed that autophagy generates Aβ peptides. They also provided evidence that in AD, autophagy is upregulated and impaired in late steps of the pathway involving clearance of partially digested material. Autophagy goes into overdrive not only in AD but also in Parkinson and Huntington diseases—presumably to handle the buildup of toxic protein aggregates in these and other neurodegenerative disorders. In line with this idea, beclin 1 levels rise after traumatic brain injury (Erlich et al., 2006).

Based on studies by Nixon and others, the prevailing thought in the AD field has been that too much autophagy leads to more Aβ. So one might predict that mice with reduced levels of beclin 1, which plays a key role in activating the autophagic pathway, might actually make less Aβ. “But we say that a deficiency in beclin 1 is also bad,” Wyss-Coray told ARF.

In the new study, co-author Small looked at brain samples from AD patients, and showed that beclin 1 mRNA levels in the entorhinal cortex (EC), which is vulnerable in AD, were reduced about 50 percent relative to non-demented subjects. RNA levels were normalized to those in the dentate gyrus, a region usually spared of AD-related damage.

At the protein level, Pickford and colleagues looked in autopsy-confirmed brains of people with AD, mild cognitive impairment (MCI), Lewy body variants of AD (LBV), or Huntington disease (HD) and compared them with age-matched healthy controls. Beclin 1 levels dropped in AD patients to 30 percent of that seen in controls and, in MCI patients, to 70 percent of control levels, the researchers found. On the whole, beclin 1 protein levels were unchanged in LBV and HD patients relative to controls. Some could argue that AD and MCI patients had lower beclin 1 protein levels because their neurons were dying. The authors discount this claim with Western data showing that levels of the neuronal marker neuron-specific enolase (NSE) remained constant among all samples regardless of disease condition.

To address whether beclin 1 reduction contributes to AD or simply occurs as a result of AD pathology, the scientists looked at beclin 1 protein levels in two different lines of very old (24 and 34 months) transgenic mice expressing high levels of mutant human APP. Compared with non-transgenic controls, beclin 1 levels were not reduced in either transgenic line, leaving open the possibility that beclin 1 reduction might occur upstream of AD pathology in the disease process.

In histological studies with APP transgenic mice on a beclin 1 heterozygote background (APP+Becn+/-), extracellular Aβ deposits were found to be nearly double that of littermate controls. Using differently aged mice with varying levels of AD pathology, the researchers showed that beclin 1 protein levels in the neocortex correlated inversely with soluble Aβ levels. Intracellular Aβ was also increased in APP+Becn+/- mice, co-localizing in part with cathepsin D, a marker for lysosomes and mature autophagosomes.

As previous studies have shown that knocking out two key autophagy genes (Atg5 or Atg7) in neurons triggered striking neurodegeneration (see Hara et al., 2006; Komatsu et al., 2006; ARF related news story), the authors wondered whether beclin 1 deficiency would exacerbate the neurodegeneration in AD mice. The answer was a resounding yes, as demonstrated in electron microscopy experiments by co-author Masliah and colleagues. The researchers saw starkly decreased immunoreactivity with three markers of synaptic integrity (synaptophysin, MAP-2, and calbindin) in APP+Becn+/- frontal cortical neurons, and even in Becn+/- mice without human APP overexpression. Electron microscopy of APP+Becn+/- neurons also revealed accumulation of abnormal, enlarged lysosomes—a feature not seen in APP+ mice with wild-type beclin 1 expression—as well as a striking array of intracellular structural abnormalities.

Finally, the authors showed that intracellular and extracellular Aβ could be reduced about twofold with beclin 1-expressing lentivirus injected into the frontal cortex and hippocampus of APP+ mice. This finding seems to jibe with previous work showing that stimulating autophagy can delay Huntington’s (see ARF related news story and ARF news story).

While the JCI work has further established the relevance of autophagy to AD, it has also raised a flurry of new questions about the cell biology of autophagic-lysosomal trafficking. Sorting out the what, how, and why behind the enlarged lysosomes in beclin-deficient neurons is one subject of ongoing investigation, Wyss-Coray told ARF. “Are they getting too much material from the autophagosomal pathway? Or is it that some of the proteins necessary for degradation are delivered through phagosomes, and maybe there’s a defect there?” he asked. “Lysosomes ultimately need to degrade what autophagosomes deliver. If they're not functioning, then you have more material accumulating in the cells.”

Even as the underlying molecular mechanisms remain mysterious, “the finding that beclin 1 overexpression reduces both the intracellular level and the extracellular deposition of Aβ in a mouse model of AD is exciting,” write Jin-A Lee and Fen-Biao Gao, University of California at San Francisco, in a commentary accompanying the JCI paper. “If this finding can be further confirmed by others in different cellular and animal models of AD, manipulating beclin 1 activity in combination with other interventions may be an attractive therapeutic approach for AD patients.”—Esther Landhuis

The Pickford et al. study adds strong support to an emerging view that autophagic-lysosomal impairment in AD can contribute to Aβ pathology and also to neurodegeneration through additional Aβ-independent mechanisms, which might be shared by other neurological diseases across the lifespan (1). The deficiencies Pickford and colleagues identified in the initial “sequestration” stages of autophagy compound other defects. We previously reported the clearance of Aβ-generating autophagic vacuoles that lead to vacuole accumulation—even in the presence of possibly slowed autophagosome formation as implied by the current findings.

Protein/vesicular trafficking defects in AD tend to be viewed from the focused perspective of how APP metabolism is altered, but, as this and other recent studies imply, the trafficking/handling of many proteins is affected by alterations of endosomes, autophagic compartments, and lysosomes, which are increasingly being linked to AD-related genetic factors (e.g., presenilin, SorLA, APP duplication, etc.). These more global effects on neuronal function are the "elephant in the room" in most current discussions of altered protein/vesicular trafficking in AD and deserve consideration as factors relevant to AD pathogenesis in their own right. In this regard, the endosomal-autophagic lysosomal system dysfunction being recognized in a growing number of other neurodegenerative diseases may well inform us about the pathogenic significance of such impairments in AD.

Another Herpes Virus-Alzheimer’s Disease Connection: Beclin Beckons
This study not only strengthens the link between AD and autophagy by relating it to a reduced beclin 1 activity in the diseased brain. It also strengthens, indirectly, another link which we proposed (1), namely, among herpes simplex virus type 1 (HSV1), autophagy, and AD—thus extending the striking HSV1-amyloid connection that we recently discovered (2). HSV1 infects, and then resides lifelong, in the peripheral nervous system (PNS) of most humans in a latent state and is reactivated periodically by events such as stress; it then causes damage—cold sores—in some of those infected.

We detected HSV1 DNA some 18 years ago in the brain of many elderly humans (3), and subsequently showed that in brain, as in the PNS, it reactivates from latency (4), possibly recurrently, triggered presumably by stress, systemic infection, etc. Further, we found that HSV1 in ApoE-ε4 carriers’ brains conferred a strong risk of AD (5), and we suggested that brain damage caused on viral reactivation was greater in ApoE-ε4 carriers, leading to the development of AD (5,6). Since then, several studies by others have linked HSV1 to AD, detecting a close homology between a sequence in Aβ and one of the viral glycoproteins (7), and showing that APP associates with HSV1 during axonal transport (8). Also, recent studies have indicated that ApoE affects HSV1 (9-12), determining its transport in tissues and its expression. (Indeed, we have found that ApoE influences outcome of infection by several other viruses, including occurrence of cold sores in ApoE-ε4 carriers—paralleling the CNS situation—see, e.g., [6].)

Our recent data reveal that HSV1 infection of cultured cells causes a large accumulation of Aβ (2) and also AD-like tau phosphorylation, and infection of mice causes Aβ accumulation in brain.

Our recent hypothesis proposes that excess Aβ produced by HSV1 action is inadequately removed by autophagy because of viral hindrance, thus allowing plaque formation to occur (1). This was based on the fact that cells infected by HSV1 attempt to demolish the intruder by an autophagic process called xenophagy, but to evade this, the viral-encoded neurovirulence protein, ICP34.5, binds to beclin and inhibits its autophagic function (13). This ability to overcome xenophagy is shared by various other infectious agents, e.g., HIV (14) (and some microbes even use autophagy for their own advantage [15,16]), but HSV1 is the only pathogen (or indeed inflammation-producing agent) detected so far in many normal elderly human brains, and is therefore the only agent in a position to cause AD-like damage—as indeed we have shown it to do in cells and mice. In fact, one of the authors of the present study, too, has discussed the possibility that viral inhibition of autophagy might contribute to “non-infectious” neurodegenerative diseases such as AD (17).